PROJECT SUMMARY This project will investigate mammalian DNA polymerase ? (Pol ?), the defining enzyme for repair of DNA double-strand breaks by polymerase theta-mediated end joining (TMEJ). This is Project 4 (?Single-molecule studies of TMEJ?) which is part of a Program Project titled, ?Polymerase theta, genome instability, and cancer?. Despite the biological importance of TMEJ, we know surprisingly little about its molecular mechanism and how defects in the process confer specific vulnerabilities in tumors. Pol ? is a large protein (290 kDa in mammalian cells) with a distinctive arrangement of a DNA polymerase domain, a helicase-like domain, and a connecting central domain. This project aims to fill several fundamental gaps in our knowledge of TMEJ, and explore novel hypotheses by employing an array of innovative biochemical, cellular, and single-molecule techniques and assays to define the key steps and molecular mechanisms of TMEJ. First, we will focus on the initial elusive steps of TMEJ including synapsis and DNA microhomology search process, and how it is modulated by other repair factors. Second, we will define the kinetics and regulation of TMEJ during cellular DSB repair. Third, we will establish how TMEJ contributes to repair of collapsed replication forks and repair of replication conflicts at secondary DNA structures. In Aim 1 ?Mechanism of TMEJ synapsis via biochemically reconstituted system? we will establish the specific contributions of Pol ? helicase, polymerase and other structural domains for initial strand pairing activity, micro-homology search, and crosstalk with NHEJ and HR In Aim 2, ?Interplay of TMEJ with NHEJ at DSB sites in cells?, we will measure the specific modes of recruitment-exclusion and organization of TMEJ repair intermediates as a function of DDR and canonical DSB repair, and how they are affected by key repair deficiencies. In Aim 3, ?TMEJ role(s) in repair of collapsed replication forks?, we will investigate the roles of Pol ? in repair of single-ended DSBs (seDSB) formed at collapsed replication forks and resoltuon of toxic secondary structures. The research work will be highly coordinated within the Program Project with the other three Projects and the three Cores. Our combined diverse approaches include molecular biology, biochemistry, structural biology, and biophysics. Substrates, proteins, and experiments will be designed with Projects 1, 2, and 3, and will be constantly monitored with feedback via Core A. Protein purification will be supported by Core B, and cell line construction by Core C.